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Development 130, 3503-3514 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00538
Wnt signalling regulates myogenic differentiation in the developing avian wing Kelly Anakwe1,*, Lesley Robson2,*, Julia Hadley1, Paul Buxton1, Vicki Church1, Steve Allen1, Christine Hartmann3,‡, Brian Harfe3, Tsutomu Nohno4, Anthony M. C. Brown5, Darrell J. R. Evans6 and Philippa Francis-West1,§ 1Department 2Department 3Department 4Department 5Department
of Craniofacial Development, King’s College, London SE1 9RT, UK of Neuroscience, Bart’s and The London, Queen Mary’s School of Medicine and Dentistry, London E1 4NS, UK of Genetics, Harvard Medical School, Boston, MA 02115, USA of Molecular Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, Japan of Cell and Developmental Biology, Weill Medical College of Cornell University and Strang Cancer Prevention Center, New York, NY 10021, USA 6School of Biosciences, Cardiff University, Cardiff CF10 3US, UK *These authors contributed equally to this work ‡Present address: Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria §Author for correspondence (e-mail:
[email protected])
Accepted 8 April 2003
SUMMARY The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the
opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and ∆Lef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.
INTRODUCTION
the subectodermal mesenchyme (Christ and Ordahl, 1995; Amthor et al., 1998). The onset of myogenic differentiation is repressed by a number of growth factors, allowing the expansion of the premyogenic pool and ultimately the number of terminally differentiated myoblasts within the limb bud. These repressive signals include scatter factor, which is expressed by the mesenchyme, and fibroblast growth factors (FGFs), which are expressed by the apical ectodermal ridge and ectoderm. In addition, bone morphogenetic protein (BMP) signalling from both the ectoderm and mesenchyme plays a repressive role, as demonstrated by the ability of the BMPs to maintain Pax3 expression in the developing limb bud (Amthor et al., 1998; Scaal et al., 1999; Edom-Vovard et al., 2001). Sonic hedgehog (Shh) also maintains the ventral muscle precursors in an
The limb myogenic progenitors arise from the ventrolateral dermomyotome of the somite in response to signals from the adjacent lateral plate mesoderm (reviewed by Buckingham et al., 2003; Francis-West et al., 2003). Following delamination, the premyogenic cells, which express the transcription factors Pax3, Lbx1 and Msx1, migrate towards the distal tip of the limb bud, where they become committed to myogenic differentiation, as shown by the onset of the expression of the myogenic determination helix-loop-helix factors, MyoD and Myf5 (reviewed by Buckingham, 2003; Francis-West et al., 2003). The premyogenic (Pax3 expressing) and early myogenic cells (MyoD/Myf5 expressing) form the pre-muscle masses, which are loose collections of cells scattered within
Key words: Wnt, Limb, Myogenic differentiation, Fibre type, Chick
3504 K. Anakwe and others undifferentiated state, possibly acting via the maintenance of BMP expression (Duprez et al., 1998; Krüger et al., 2001; Bren-Mattison and Olwin, 2002). It is not yet totally clear whether the onset of myogenic differentiation within the limb bud is just a default or passive state following the release of the premyogenic cells from their inhibitory cues, or whether positive inductive factors are needed. However, recent work has suggested that inductive signals from the FGF family are required for differentiation (Marics et al., 2002). Thus, FGF signalling is initially repressive but is later inductive or permissive for myogenic differentiation, emphasizing the complexity of the molecular regulatory network that controls myogenesis in the limb bud. Myoblasts subsequently coalesce to form the dorsal and ventral muscle masses, which are the template of the future muscles (Schramm and Solursh, 1990). Myoblasts also start to differentiate terminally by switching on the expression of the terminal differentiation factors, the myosin heavy chains (MyHCs). These terminally differentiated myoblasts then fuse, forming multinucleated fibres that can contract (Hilfer et al., 1973; Sweeney et al., 1989). This period of primary fibre development is followed by secondary fibre formation. The secondary fibres align on the surface of the primary fibres, starting at day 7 in the chick embryo, and grow to constitute the bulk of skeletal muscle at birth (Fredette and Landmesser, 1991). Each muscle is characterized by a unique profile of slow and fast fibre types that will determine how that muscle will function (Miller and Stockdale, 1986a; Miller and Stockdale, 1986b). Fast fibres express one of the fast MyHC isoforms and usually use glycolytic metabolism. They can generate high force but fatigue easily. By contrast, slow fibres use oxidative metabolism and express slow isoforms of the MyHC (Hughes and Salinas, 1999). These fibres contract slowly and are able to maintain a contraction for longer without fatigue. When and where fibre-type commitment occurs has been a running debate. A recent elegant study in which the somitic precursors of the quail pectoralis muscle were grafted into the equivalent position in a chick host suggested that commitment occurs within the somite (Nikovits et al., 2001). In these studies, the slow/fast patterning of the pectoralis muscle was characteristic of the donor and not the host. Clonal analysis studies have also shown that myogenic cells are heterogeneous in their slow/fast MyHC expression and are committed to their different fibre-type fates by stage 24/25 in the quail (DiMario et al., 1993) (reviewed by Stockdale, 1990). This is in contrast to fate-labelling studies in which individual premyogenic clones were marked with a specific nucleotide tag (Kardon et al., 2002). These studies showed that a single premyogenic cell could give rise to both slow and fast myoblasts in addition to a distinct lineage (endothelial cells). These latter data suggest that environmental cues, presumably within the limb bud, control fibre-type patterning and are consistent with other data in which clones of foetal or satellite myogenic cells were shown to differentiate or to modify their fate when grafted into a new host (Hughes and Blau, 1992; DiMario and Stockdale, 1997; Robson and Hughes, 1999). One way of reconciling this data is to argue that different muscles in the limb can be governed by a different set of signalling interactions. An alternative, and equally plausible, argument is that the premyogenic cells are biased to one fibre-type fate as they leave the somite but that they exhibit plasticity (i.e. that they
are not committed) and their ultimate fate is determined or modified by local environmental signals (reviewed by FrancisWest et al., 2003). Factors that specify limb myogenic fibre-type differentiation are unknown. In chick somites and zebrafish adaxial musculature, Shh or hedgehog signalling promotes and is essential for slow fibre-type formation. Therefore, loss of Shh signalling inhibits slow fibre development, whereas excess Shh promotes slow fibre formation (Currie and Ingham, 1996; Blagden et al., 1997; Cann et al., 1999; Lewis et al., 1999; Barresi et al., 2000). However, in the limb bud, Shh does not appear to determine myogenic cell fate but does initially prevent differentiation of a subpopulation of the presumptive slow muscle precursors, maintaining them in a proliferative state and, ultimately, increasing the number of slow fibres (Bren-Mattison and Olwin, 2002). The role of the Wnt family of secreted factors during limb myogenic development has to date been neglected, yet members of this family initiate myogenic differentiation in the epaxial and hypaxial musculature, substituting for the neural tube and ectodermal signals, respectively (Ikeya and Takada, 1998; Cossu et al., 1996; Tajbakhsh et al., 1998). In addition, overexpression of the Wnt antagonist Sfrp3 blocks myogenic differentiation in mouse somites (Borello et al., 1999). The Wnt family consists of 19 members, which can act through one of three pathways that might depend on the Frizzled receptor profile of the receiving cell – first, through the classical βcatenin pathway, second, through a calcium protein kinase C (PKC)-mediated pathway and, finally, through a novel Jun kinase pathway (reviewed by Church and Francis-West, 2002). Several members of this family are expressed in the limb, where they control patterning, outgrowth and/or differentiation (reviewed by Church and Francis-West, 2002). Wnt5a, Wnt11 and Wnt14 are expressed in the mesenchyme, whereas Wnt4, Wnt6 and Wnt7a are expressed in the ectoderm, the last of these being restricted to the dorsal surface, where it controls dorsal ventral patterning (reviewed by Church and Francis-West, 2002). In addition, Wnt3a is expressed in the apical ectodermal ridge (AER). Therefore, within the limb bud the pre- and differentiating myogenic cells are within range of Wnt signalling, which is thought to propagate over 11-12 cell diameters, from the ectoderm and mesenchyme. Thus, it is possible that, as in the somites, Wnts might regulate myogenic differentiation. Finally, Sfrp2 is expressed in the migrating muscle precursors in the chick, whereas Sfrp1 is expressed in the lateral dermomyotome in the mouse, again suggesting that modulation of Wnt signals might control limb myogenic differentiation (Ladher et al., 2000b; Lee et al., 2000). Here, we show by gain- and loss-of-function studies that different members of the Wnt family have distinct effects on limb muscle development, controlling the number of terminally differentiated cells and the number expressing either slow or fast MyHCs. Thus, we identify novel functions of Wnt signalling during limb myogenic differentiation. MATERIALS AND METHODS Embryos Fertilized Ross White chicken eggs were supplied by Poyndon Farm (Goff’s Oak, UK) or SPF-free eggs were obtained from Lohman
Wnts and limb muscle development 3505 Tierzucht, Germany. The eggs were incubated at 38±1°C and the embryos were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1951). In situ hybridization In situ hybridization to whole embryos was carried out as described by Francis-West et al. (Francis-West et al., 1995). cDNA and ribroprobes were made as described previously: MyoD (Lin et al., 1989), cWnt5a (Kawakami et al., 1999) and cWnt11 (Tanda et al., 1995). Retroviral constructs and culture Concentrated retroviral stocks and retrovirally infected chicken embryonic cells for grafting were prepared as described by Logan and Francis-West (Logan and Francis-West, 1999). The Wnt3a, Wnt5a, Wnt7a, Wnt14, activated β-catenin, Sfrp2 and dominant-negative Lef1 (∆Lef1) retroviruses are as described previously: Wnt3a, activated β-catenin and ∆Lef1 (Kengaku et al., 1998), Wnt5a (Kawakami et al., 1999), Wnt7a (Rudnicki and Brown, 1997), Wnt14 (Hartmann and Tabin, 2001) and Sfrp2 (Ellies et al., 2000). The other retroviruses were constructed in RCAS(BP) and encode Xenopus Wnt4, mouse Wnt6, a partial chick Wnt11 cDNA equivalent to the Xenopus Wnt11 construct described by Tada and Smith (Tada and Smith, 2000), which acts as a dominant-negative, activated rat calmodulin kinase II (Kühl et al., 2000a), Xenopus Dsh, which lacks the PDZ domain (Dsh∆PDZ) (Tada and Smith, 2000), enhanced-green fluorescent protein (eGFP; Clontech), or in RCAS-L14, which encodes chicken Wnt11 (Tanda et al., 1995).
myogenic differentiation, we analysed the expression of Wnts by whole-mount in situ hybridization and compared their expression, both temporally and spatially, to that of the muscle determination factor MyoD. As the limb myogenic precursors enter and differentiate in the limb bud, they come into contact with the mesenchymal signals Wnt5a and Wnt11. Wnt5a is initially expressed throughout the mesenchyme at stage 18, later becoming predominantly confined to the distal tip with lower expression levels proximally (Fig. 1A-C,J-M and data not shown) (see also Dealy et al., 1993; Kawakami et al., 1999). Between stages 25 and 27, Wnt5a expression is also found in the central core next to the developing cartilage elements and muscle masses (Fig. 1C,M). By contrast, Wnt11 is not expressed until after the onset of MyoD expression and hence myogenic commitment (Fig. 1D,G). Wnt11 is first expressed in the proximal dorsal subectodermal mesenchyme
Retroviral misexpression studies Grafting of retrovirally infected cells into stage 18-21 limb buds was as described in Francis-West et al. (Francis-West et al., 1999). Stage 19/20 and 21/22 wing bud micromass cultures were prepared as described in Francis-West et al. (Francis-West et al., 1999) except that they were plated in the presence of high titre (>108 pfu) RCAS(BP) retroviruses and were cultured in the absence of ascorbate. The micromasses were cultured for three days. Immunohistochemistry Embryos were dissected and placed into 20% sucrose in PBS at 4°C. They were embedded in OCT compound (BDH Lab Supplies) and cryosectioned at 15 µm. Micromass cultures were fixed in methanol for 2 minutes and were washed twice for 5 minutes with PBS. Muscle development was analysed using the following primary antibodies diluted in PBS: A4.1025 (1 in 100), which recognizes all terminally differentiated muscle cells and A4.840 (1 in 50), which recognizes cells expressing the slow MyHC isoforms SM3 and SM1 (from the developmental hybridoma bank) (Webster et al., 1988; Hughes and Blau, 1992). The Pax3 antibody (1 in 100) was a gift from C. Ordahl, C. Marcelle and M. Bronner-Fraser, and is described by Baker et al. (Baker et al., 1999). The GAG antibody (1 in 5) is as described in Logan and Francis-West (Logan and Francis-West, 1999). Incubation with the primary antibodies was followed by incubation with horse anti-mouse IgG (γ specific) conjugated to FITC (Vector; 1:400) and donkey anti-mouse IgM (µ specific) conjugated to Cy3 (Jackson; 1:800) for at least 1 hour at room temperature. Cultures and sections were mounted under coverslips with PBS:glycerol (1:9) with 0.1% phenylenediamine as an antifade reagent. They were then viewed and the images were captured using a Leica DMRD microscope and the HiPic32 program. The data was analysed using Student’s t test.
RESULTS Correlation of Wnt expression with myogenic differentiation To determine potential roles of Wnt signalling during limb
Fig. 1. Correlation of Wnt5a and Wnt11 expression with myogenic differentiation. The expression of Wnt5a (A-C,J-M), MyoD (D-F,N) and Wnt11 (G-I,N) in stage 22 (A,D,G), 25 (B,E,H) and 27 (C,F,I) wing buds was determined by whole-mount in situ hybridization. (J-N) Transverse vibratome sections of wing buds expressing Wnt5a (J-M) and Wnt11 and MyoD (N) at stages 22 (J,K), 26 (N) and 27 (L,M). In (A-M), expression is shown in purple/red. In (N), MyoD expression is shown in red and Wnt11 expression is shown in purple. In (A-N), anterior is uppermost and, in (A-I), distal is to the right. In the vibratome sections, (J) is more distal than (K), and (L) is more distal than (M). In (M), developing cartilage is circled in red, whereas developing muscle is circled in black. DM, dorsal muscle mass; R, radius; U, ulna; VM, ventral muscle mass. Scale bars: 100 µm in A,D,G; 150 µm in B,C,E,F,H,I; 125 µm in J,K; 150 µm in L,M.
3506 K. Anakwe and others overlying the myogenic cells at late stage 22 in the wing (data not shown) (see Tanda et al., 1995). By stage 24, Wnt11 transcripts are clearly detectable in the dorsal mesenchyme and are also found in the ventral subectodermal mesenchyme, again overlying the developing myogenic precursors (Fig. 1E,H and data not shown). Between stages 24 and 30, Wnt11 expression extends distally over the developing myogenic cells and is slightly more advanced on the dorsal side in correlation with the advanced rate of myogenic differentiation (Fig. 1E,F,H,I,N and data not shown). At stage 26, Wnt14 is also expressed in the mesenchyme around the developing cartilage elements and forming joint regions, where it might influence primary muscle fibre development, which is not yet complete (V.C. and P.F.W., unpublished) (Hartman and Tabin, 2001). In addition to the mesenchymal signals, particularly at early stages of differentiation, the myogenic precursors also come into close proximity with ectodermal signals, which are known to influence the rate of myogenic differentiation (Amthor et al., 1998). Wnt4, Wnt6 and Wnt7a are expressed in the ectoderm, the latter being confined to the dorsal surface, whereas Wnt3a is expressed in the AER (data not shown) (reviewed in Church and Francis-West, 2002). The relationship between the myogenic cells and Wnt expression in a stage 24 wing is illustrated in Fig. 2A. In summary, the premyogenic cells are within range of Wnt signals from the ectoderm and are in contact with or in close proximity to the mesenchymal signals Wnt5a and Wnt11. Overall, the different expression of Wnt5a and Wnt11 between stages 23 and 27 correlates with the ultimate distribution of slow and fast fibres in the primary muscle fibres in the wing. In general, slow fibres are concentrated towards the centre of the limb bud, where Wnt5a transcripts are found, whereas the fast fibres are found closer to the ectoderm, where Wnt11 transcripts are located (Fig. 2B,C). Wnt signalling affects myogenic differentiation in vitro To analyse the potential roles of Wnt signalling during myogenic development, we first overexpressed these factors using the replication-competent retrovirus RCAS(BP) in an in vitro micromass culture that recapitulates myogenic differentiation in vivo (Swalla and Solursh, 1986; Archer et al., 1992). In addition, it has the advantage that the effect of Wnts
on patterning are uncoupled from their effects on myogenic development. For example, overexpression of Wnt7a dorsalizes the ventral limb, whereas Wnt3a overexpression induces ectopic AER formation and hence additional regions of outgrowth, which would induce secondary changes in muscle patterning and differentiation. Changes in muscle differentiation were assessed by double-labelling using the antibodies A4.1025, which is a pan-MyHC marker, and A4.840, which recognizes the slow MyHC SM3 and an embryonic MyHC, SM1. SM1 is initially expressed by most myogenic cells but is downregulated rapidly in fast myogenic cells while being maintained in slow-MyHC-expressing cells (Webster et al., 1988; Hughes and Blau, 1992). The A4.840 antibody will recognize slow MyHCs present in mixed slow/fast fibres and fibres that exclusively express slow MyHC; for ease of reading, these myogenic cells will be referred to as ‘slow’. To determine the number of myoblasts expressing exclusively fast MyHCs, the number of myogenic cells recognized by the antibody A4.840 was subtracted from the number recognized by the pan-A4.1025 antibody. For each Wnt, at least three independent experiments were carried out, consisting of at least three micromass cultures. Control micromasses were infected with a retrovirus encoding green fluorescent protein (GFP). Control cultures typically possessed 1060±64 MyHCexpressing cells, of which 94% were mononucleate. As for the control micromass cultures, 92% or greater of MyHCexpressing cells in Wnt-infected cultures were mononucleate (Wnt3a, 100%; Wnt4, 92%; Wnt5a, 96%; Wnt6, 94%; Wnt7a, 99%; Wnt11, 92%; Wnt14, 97%). Overexpression of different members of the Wnt gene family had two distinct effects on myogenic differentiation. First, Wnt signalling could change the number of terminally differentiated myogenic cells. Second, a change in the number of slow and/or fast-MyHCexpressing cells was observed (Figs 3, 4, Table 1). Wnt5a and Wnt6 had no significant effect on the number of myocytes, whereas Wnt3a significantly decreased the number of terminally differentiated myogenic cells (Fig. 3A,B,D,E, Fig. 4, Table 1). By contrast, Wnt4, Wnt7a, Wnt11 and Wnt14 overexpression increased the number of MyHC-expressing cells (Fig. 3A,C,F-H, Fig. 4, Table 1). These changes in number were linked with different changes in the number of fast and/or slow myocytes. In Wnt3a transfected cultures, the
Fig. 2. Correlation of Wnt expression with muscle differentiation. (A) A sketch through the dorso-ventral axis of a stage 24 wing bud showing the relationship between Wnt expression and the developing myogenic cells. Initially, Wnt7a is expressed throughout the dorsal ectoderm but, by stage 24, its expression is restricted to the dorsal ectoderm overlying the progress zone. (B) Transverse section through an 8-day limb showing the expression of slow MyHC (orange) versus fast MyHC (green). (C) Diagrammatic sketch of (B). ANC, anconeus; EDC, extensor digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; Ent, entepicondyloulnaris; FCU, flexor carpi ulnaris; PP, pronator profundus; PS, pronator superficialis; SUP, supinator.
Wnts and limb muscle development 3507
Fig. 3. Effects of Wnt overexpression on fibre-type differentiation in vitro. (A-P) Fluorescent images of stage 21/22 wing micromass cultures showing terminally differentiated myogenic cells that have been visualized with antibody A4.1025, which recognizes both slow and fast MyHCs (A-H, green), and antibody A4.840, which specifically recognizes slow MyHCs (I-P, red). The micromass cultures have been infected with control RCAS(BP) virus (A,I) or retroviruses expressing Wnt3a (B,J), Wnt4 (C,K), Wnt5a (D,L), Wnt6 (E,M), Wnt7a (F,N) Wnt11 (G,O) or Wnt14 (H,P). Scale bars, 100 µm.
Table 1. Summary of the effect of Wnt signalling on limb myogenic differentiation Protein
Fig. 4. The effects of Wnt overexpression on fibre-type differentiation in vitro. The bar chart shows the total number of differentiated myogenic cells, and the number expressing fast or slow MyHCs in 3-day limb micromass cultures that have been infected with either a control RCAS(BP) virus or retroviruses expressing Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7a, Wnt11 or Wnt14, as shown in Fig. 2. The slow population of myoblasts (red) might express either exclusively slow MyHC or both slow and fast MyHCs, whereas the fast myogenic population (yellow) only expresses fast MyHC isoforms. *, P
